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Biomedical subjects

F Jakob

Publications and source records attributed to F Jakob.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression and regulation of aromatase cytochrome P450 in THP 1 human myeloid leukaemia cells.

Aromatase cytochrome P450 mRNA and activity was strongly expressed in THP 1 myeloid leukaemia cells after treatment with phorbol-myristate-acetate (PMA) and dexamethasone, low level expression was caused by calcitriol. mRNA species of 4.0, 3.0, 2.4 and 1.1 kb size were differentially stimulated. After calcitriol-mediated differentiation (72 h, measured by CD 14 expression) mRNA expression was further enhanced by PMA (45-fold), dexamethasone (15-fold), oestradiol (3.7-fold), testosterone (2.5-fold) and androstenedione (3.5-fold). Forskolin, cAMP and follicle stimulating hormone had no stimulatory effect. Oestradiol formation from testosterone (oestradiol radioimmunoassay in culture supernatants) increased to > 2000 pg/ml/10(6) cells/24 h after PMA-stimulation, mirrored mRNA expression and was suppressed below 10% of original values in the presence of 4-OH-androstenedione. Exons I.2 and I.4 were expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. A new splicing variant was expressed after calcitriol-stimulation, which did not hybridize to an exon II-derived oligonucleotide but to an exon III-derived one. Local aromatisation of androgens into oestradiol may be important in the concerted crosstalk of cells of the monocyte/macrophage lineage with their respective tissues in inflammation and bone metabolism.

Androstenedione↗
PubMed1995

Neuroendocrine support for a relationship between "novelty seeking" and dopaminergic function in alcohol-dependent men.

The personality traits "novelty seeking", "harm avoidance", and "reward dependence" were rated using Cloninger's Tridimensional Personality Questionnaire in 20 male inpatients, suffering from moderate to severe alcohol dependence (ICD-10, DSM-III-R). The same individuals' dopamine receptor sensitivity was determined by stimulating a neuroendocrine response with a dopamine receptor agonist (apomorphine). The amount of growth hormone released was measured and taken as a biological parameter for the sensitivity of D2 dopamine receptors located in the hypothalamus. Our data indicate that in abstinent alcohol-dependent men no statistically significant correlation exists between a person's apomorphine-induced growth hormone release and his "harm avoidance" or "reward dependence" score. On the other hand, a significant correlation (r = .47; p = .035) was found between growth hormone release and the person's "novelty seeking" score. This result supports Cloninger's hypothesis by giving neuroendocrine evidence that the personality dimension "novelty seeking" is related to dopaminergic activity in alcohol-dependent men.

Adult↗
PubMed1995

Alcohol dependence, family history, and D2 dopamine receptor function as neuroendocrinologically assessed with apomorphine.

Fifteen alcohol dependent men with an alcohol dependent first degree relative (i.e. family history positive or FHP), 15 well matched alcohol dependent men without a family history for alcohol dependence (i.e. family history negative or FHN), and 15 healthy controls (CONTR) participated in this study. The three groups were compared according to their postsynaptic D2 dopamine receptor function as assessed by growth hormone release after stimulation with the dopamine receptor agonist apomorphine. Statistical evaluation was done by planned comparisons within a one-way ANOVA. Alcohol dependent subjects significantly differed from CONTRs as long as family history was not taken into account (t(42) = 2.38; P = 0.022*). When differentiating according to family history, both FHPs and FHNs maintained a blunted growth hormone response. However, the difference between FHNs and CONTRs, though present, dropped out of statistical significance (t(42) = 1.65; P = 0.105); at the same time, the difference between FHPs and CONTRs became slightly stronger (t(42) = 2.47; p = 0.017*). In conclusion, our data give neuroendocrinological support to the assumption that a reduced D2 dopamine receptor function in alcohol dependent men is not only a state marker of residual heavy drinking but also a genetically determined trait marker.

Adult↗
PubMed1995

Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells.

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.

Actins↗
PubMed1994

Topoisomerase I-inhibition enhances vitamin D-responsive expression of the receptor for lipopolysaccharide binding protein CD 14.

Pretreatment (4h) of human HL 60 leukemia cells with the topoisomerase I-inhibitor camptothecin (7-28 x 10(-9) Mol/l, single dose) enhanced vitamin D3-responsive CD 14 expression (10(-11)-10(-8) Mol/l vitamin D3) up to twofold, as measured by fluorescence activated flow cytometry after 1-3 days. Camptothecin by itself caused marginal effects. Hybridization of total RNA with oligonucleotides (bases 1358-1333 of CD 14 sequence) showed increased steady state levels of the 1.75 kb CD 14 mRNA after 24 h when normalized to beta-actin. The nuclear accumulation of [3H]-vitamin D3/receptor complexes (VDR) in a nuclear translocation assay was significantly enhanced (by 3 h) in the presence of camptothecin. We conclude that inhibition of topoisomerase I enhances vitamin D3-stimulated CD 14 expression. Topoisomerase I might either be a negatively active transcription factor itself or maintenance of DNA-bending, local supercoiling and accessibility of responsive elements might lead to reduction of VDR turnover and produce a stimulatory effect on vitamin D3-responsive transcription.

Acute-Phase Proteins↗
PubMed1993

[Unilateral autonomous aldosterone production in hyperaldosteronism suppressible by dexamethasone].

A 21-year-old woman with weight loss, palpitations and facial flush was found to have hypertension (up to 200/130 mm Hg) and mild hyperkalaemia (3.4 mmol/l). Extensive diagnostic tests revealed hyperaldosteronism with contrast storing in the right adrenal gland on scintigraphy after injection of dexamethasone (2 mg daily for one week). The hyperaldosteronism could not be suppressed by dexamethasone. Analysis of venous blood separately from each side pointed to aldosterone production in the right adrenal (right renal vein: 80 ng/dl, drainage area of the right adrenal vein: 114 ng/dl, left renal vein: too low to measure). The right adrenal gland was removed. No adenoma was found histologically. After the operation the aldosterone level was reduced and the blood pressure transiently fell. But both had risen again after 3 months. Renewed tests revealed dexamethasone-remediable hyperaldosteronism. On treatment with hydrocortisone (15-5-5 mg) and 50 mg metoprolol the patient became normotensive without any other medication.

Adrenal Cortex Hormones↗
PubMed1993

Characterization of five monoclonal antibodies raised against domain E of the porcine estradiol receptor.

Male Balb C mice were immunized with a pure 32 kDa fragment of the porcine estradiol receptor spanning the domain E. Five antibody-producing hybridoma lines were established from fusions of spleen cells with the myeloma lines SPO and NSO. The antibodies were analyzed for interaction with native and with denatured intact receptor and receptor fragments, respectively. The antigenic determinant for antibody 13H2 is a native epitope which retains its combining activity after receptor denaturation. The antibodies 1F5, 1G5, 3E6 and 2H10 react with denatured proteins only. The presence of the determinants of all five antibodies has also been demonstrated in human, bovine, rat and mouse estradiol receptor.

Animals↗
PubMed1992

Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes--coincident rise of DNA-relaxing activity in nuclear extracts.

High affinity receptors (VDR) for 1,25-dihydroxycholecalciferol (calcitriol) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes (PBL). HL60 cells, expressing some characteristics of promyelocytes, can be induced to monocytoid differentiation by calcitriol. Specific nuclear translocation of [3H]calcitriol/VDR was examined after exposure of whole cells to 10(-9) M/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei. Specific nuclear translocation of [3H]calcitriol/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites/nucleus after 3 h of incubation in HL60 cells, whereas a maximum of approximately 310 binding sites/nucleus was found after 3 h in PBL. Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h. Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse (pulse/chase-experiments). No difference of VDR retention in pulse and pulse/chase-experiments was seen in PBL, where VDR halflife was approximately 30 min. No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in [3H]calcitriol. Radiolabeled hormone/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone--in contrast to identical experiments with intact cells--did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound. The activity of DNA relaxing enzymes (e.g. topoisomerases I and II) in nuclear extracts was measured using a PBR 322-relaxation-assay. Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol (final ethanol concentration 0.0001% v/v) in HL60 and PBL. The enhanced activity disappeared after 2 h in PBL, whereas it was still enhanced by 4 h in HL60. No effect was seen in ethanol treated controls. We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined, most likely due to translocation of receptor proteins after hormone binding. Translocated hormone/receptor complexes compete for a limited number of specific nuclear binding sites. Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment, thus initiating DNA-unwinding, a prerequisite of transcription initiation.

Biological Transport↗
PubMed1992

Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro.

Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0-25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agar-electrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations.

Blotting, Western↗
PubMed1992

Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.

Animals↗
PubMed1991

The proton-driven dissociation of oestradiol-receptor dimers as a preparative tool. Isolation of a 32 kDa fragment from porcine uteri and assignment of C-terminal origin by partial sequencing.

Homodimers of the porcine oestradiol receptor dissociated at pH 6.4. The monomers reassociate after neutralization. This property is retained in a 32 kDa receptor fragment generated by co-adsorbed endopeptidases from cytosolic receptor bound to heparin-Sepharose. The fragment was purified by successive gel filtrations in the dimer and monomer states. Precipitations with ethanol and (NH4)2SO4 respectively served as concentrating steps. In all, 10-15 nmol of the homogeneous fragment were recovered from 8 kg batches of porcine uteri with a approximately 10(5)-fold enrichment and in approximately 20% yields. Its oestradiol-binding capacity was identical with that of the intact receptor. The N-terminus was blocked. Two decapeptides from a tryptic digest were sequenced. One of them corresponded to amino acids 353-362 of the human receptor, a sequence fully conserved in all species investigated. The second peptide differed in positions 553, 554 and 557 from the 549-558 sequence of the human protein.

Amino Acid Sequence↗
PubMed1986

[Effective treatment of peritoneal carcinosis by intraperitoneal cis-diamminedichloroplatinum (c-DDP) administration with systemic sodium thiosulfate protection. Clinical results and pharmacokinetics].

Thirteen patients with cytologically or histologically confirmed ascites from various malignancies have received 37 courses of intraperitoneal cis-platinum (cDDP). The tumor had to be confined to the peritoneal cavity or cause major symptoms by peritoneal carcinosis. cDDP (90-150 mg/m2 in 21 saline) was administered intraperitoneally with concurrent i.v. infusion of sodium thiosulfate. Courses were repeated in 4-week intervals. Total platinum was assayed by flameless atomic absorption spectrophotometry. With a dwell of 4 h 31.5 +/- 17.2% of platinum was recovered. Mean peak platinum concentration in the peritoneal cavity was 54.6 +/- 21 micrograms/ml and that in serum was 2.6 +/- 1.1 micrograms/ml. The ratio of peritoneal to serum platinum concentration dropped from 41 +/- 18.2 after 1 h to 6.9 +/- 4.7 after 5 h. The area under the curve for the peritoneum was approximately 11-fold greater than the area under the curve for serum. Within 12 h 31.7 +/- 10.7% of the administered dose was excreted in urine. Ten patients had disappearance of ascites lasting 6-135 + weeks. The treatment was generally well tolerated, no serious side effects were observed. This study demonstrates a pharmacokinetic advantage of intraperitoneal chemotherapy with cDDP and an effectiveness against peritoneal carcinosis from various malignancies with minimal systemic toxicity.

Adult↗
PubMed1979

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.

Amino Acid Sequence↗